Introduction

pIII-based phage display has been used to identify antibody fragments specific to various antigens (1,2,3,4,5). To enhance the level of the displayed antibody, Rondot et al. used a gene-III-deleted helper phage called Hyperphage to rescue the phagemid encoding antibody-pIII fusion protein (6). As a result, the number of single-chain variable fragments (scFvs) presented on the consequent phagemid particles could increase by over two orders of magnitude. However, the production of Hyperphage was so cumbersome that it was necessary to construct an Escherichia coli packaging cell line (DH5α/pIII) to supply pIII for the phage assembly in order to avoid the presence of a plasmid during helper phage production. Furthermore, the yield of Hyperphage (about 1.34 × 10⁹/mL) was rather low. Also, most phagemids used in constructing a phage display scFv library produced pIII under the control of a wild-type Lac promoter, which is not absolutely stringent, thus pIII could be produced in cells in the absence of isopropyl β-D-1-thiogalactopyranoside (IPTG) to mediate pIII resistance to the helper phage superinfection (7). Therefore, glucose, a pLac repressor, is often added to the culture medium to repress pIII expression (8).

To solve the problem, we employed a novel method of pIII-based antibody phage display by applying F⁺ Escherichia cells bearing a gene-III-lacking helper phage genome (VCSM13d3). The modified cells were named Hpd3cells, and the concept of Hpd3cells is shown in Figure 1.

Figure 1. The concept of Hpd3cells. (Click to enlarge)

Materials and Methods

Plasmid Constructs

Standard cloning procedures were carried out according to Sambrook et al. (9). VCSM13d3 was kindly provided by Dr. Jasna Rakonjac and Prof. Peter Model (10); pCANTAB5E-T was derived from pCANTAB-5E (GE Healthcare, Piscataway, NJ, USA) by inserting oligonucleotides encoding thrombin cleavage site behind NotI restriction enzyme recognition site; pET28a-EGFP was obtained by inserting enhanced green fluorescent protein (EGFP) DNA from pEGFP-N1 (Clontech Laboratories, Mountain View, CA, USA) into pET28a (EMD Biosciences, San Diego, CA, USA); and dpET-EGFP-S2 was constructed by inserting coding sequence of S2 domain of epidermal growth factor receptor (EGFR) into pET28a-EGFP.

Immunoblots

Approximately 10¹⁰ phages were loaded per lane on a 10% polyacrylamide gel. Blocking was done with 2% skim milk powder in phosphate-buffered saline (PBS) for 2 h at room temperature, and immunostaining was conducted with mouse MAb anti-m13 pIII (New England Biolabs, Ipswich, MA, USA) recognizing the C terminus of pIII coat protein of M13KO7 and goat anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP)-streptavidin conjugate (ImmuClub Labs, Sunnyvale, CA, USA), and visualized with a SuperSignal® West Pico kit (Pierce Biotechnologies, Rockford, IL, USA).

Determination of Antigen Binding Reactivity by Phage ELISA

To determine antigen binding specificity of recombinant phage particles, 100 ng bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO USA) or recombinant EGFP in coating buffer (0.1 M NaHCO₃, pH 9.1) were coated in microtiter plates at 4°C overnight. Recombinant EGFP protein was produced by growing BL21(DE3) cells harboring pET28-EGFP and affinity purifying with Ni-NTA resin (Qiagen GmbH, Hilden, Germany). The plate was blocked with 1% BSA in PBS, and 10¹⁰ scFv phages packaged with either M13KO7 or Hpd3cells in 1% BSA solution were applied to each well for 1 h at room temperature. M13KO7 10¹⁰ helper phages were used as negative controls. After being washed six times with PBS containing 0.5% Tween®-20, the bound phages were detected with anti-M13 antibody conjugated with HRP (GE Healthcare) with the signal visualized with ABTS substrate (Sigma-Aldrich) and quantified with an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Rad Laboratories, Hercules, CA, USA) at absorbance (A)₄₀₅. To observe the sensitivity of antigen binding reactivity of phage particles packaged with either M13KO7 or Hpd3cells, ELISA was performed as previously described, except that serial dilutions of purified recombinant EGFP protein were applied to the coating of microtiter plates.

Preparation of Antigen and Immunization

The recombinant protein EGFP-S2 was produced in BL21(DE3) and then affinity purified with Ni-NTA resin. With 100 µL Freud's adjuvant (Sigma-Aldrich), 100 µg EGFP-S2 proteins in 100 µL 0.9% NaCl were mixed and injected subcutaneously into each mouse. Immunization was repeated three times with the same amount of EGFP-S2 protein injected subcutaneously every 2 weeks. Two weeks following the third time of immunization, 50 µg EGFP-S2 were injected intravitreally into the mouse. Four days later, the mice were sacrificed, the spleens obtained.